Cytolytic lymphocytes (CTLs) possess cytoplasuic granules that are released in response to target cell recognition. CTL granules contain secretory proteins such as perforin and serine proteases, which are thought to contribute to the induction of target call death. Perforin has been shown to be directly cytolytic. In the presence of Ca.sup.++, it inserts into the target cell plasma membrane where it aggregates to form osmotically active ion channels {Lichtenheld, M. G., et al, (1988), "Structure and function of human perforin", Nature, 335: 448-451; Hameed, A., et al, (1989), "Cytolysis by Ca-permable transmembrane channels. Pore formation causes extensive DNA-degradation and cell lysis", J. Exp. Med., 169: 765-777}. The recent demonstration that transfection of perforin cDNA into rat basophilic leukemia (RBL) cells confers the ability to lyse erythrocytes via a regulated secretory mechanism supports a direct role for perforin in lymphocyte-mediated cytolysis {Shiver, J. W. and P. A. Henkart, (1991), "A noncytotoxic mast cell tumor line exhibits potent IgE-dependent cytotoxicity after transfection with the cytolysin/perforin gene", Cell 64: 1175-1181}. The inability of perforin-transfected RBL cells to efficiently lyse nucleated cells, however, suggests that additional granule components are required for optimal lymphocyte-mediated killing. That perforin is not the only cytolytic effector molecule is supported by the ability of natural killer (NK) cells and CTLs to kill some target cells in the absence of extracellular Ca.sup.++, which is required for perforin activity {Tirosh, R. and G. Berke, (1985), "T Lymphocyte mediated cytolysis as an excitatory process of the target. I. Evidence that the target may be the site of calcium action", Cell Immunol., 75: 113-123}. Furthermore, cytolytic lymphocytes that express little or no perforin (e.g., CD4.sup.+ CTL clones) have been shown to be potent cytolytic effector cells {Takayama, H., et al, (1991), "Antigen-specific directional target cell lysis by perforin-negative T lymphocyte clones", Inter. Immunol., 3: 1149-1156}. The results imply that perforin-independent cytolytic effector mechanisms contribute to at least some forms of target cell killing.
In addition to perforin-mediated lysis, CTLs have been shown to induce in target cells a pathway of programmed cell death known as apoptosis {Russell, J. H. (1983), "Internal disintegration model of cytotoxic lymphocyte-induced target damage", Immunol. Rev., 72: 97-118}. A convenient marker of this autolytic pathway is the fragmentation of target cell DNA into integer multiples of a 200 bp nucleosome-sized monomer {Wyllie, A. H., (1980), "Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation", Nature 284: 555-556; Duke, R. C., et al, (1983), "Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis", Proc. Natl. Acad. Sci., 80: 6361-6365}. The resulting "ladder" of DNA fragments is considered to be characteristic of this programmed suicide pathway. The observation that perforin induces cell lysis, but not DNA fragmentation {Duke, R. C., et al, (1989), "Purified perforin induces target cell lysis but not DNA fragmentation", J. Exp. Med., 170: 1451-1456} suggests that other granule components are likely to be responsible for the induction of apoptotic cell death. The granzymes, a family of granule-associated serine proteases, are candidate perforin-independent cytolytic effector molecules {Pasternack, M. S. and H. N. Eisen, (1985), "A novel serine esterase expressed by cytotoxic T lymphocytes", Nature, 314: 743-745; Masson, D. and J. Tschopp, (1987), "A family of serine esterases in lytic granules of cytolytic T lymphocytes", Cell 49: 679-685}. Although purified granzymes are not directly cytotoxic, the ability of protease inhibitors to block lymphocyte-mediated cytolysis suggests that they play a role in target cell killing {Lavie, G., et al, (1985), "The mechanism of human NK cell mediated cytotoxicity. Mode of action of surface-associated proteases in the early stages of the lytic reaction", J. Immunol., 135: 1470-1476; Rodgers, K. E., et al, (1988), "Inhibition of cytotoxic T lymphocyte and natural killer cell-mediated lysis by O,S,S-trimethyl phosphorodithioate is at an early post-recognition step", J. Immunol., 140: 564-570}. The observation that granzyme A, the most abundant granule-associated serine protease, can induce DNA fragmentation in detergent permeabilized EL4 cells argues that these molecules might contribute to the induction of apoptosis in CTL targets {Hayes, M. P., et al, (1989), "Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A", J. Exp. Med., 170: 933-946}. The further demonstration that the combination of granzymes and perforin can induce DNA fragmentation in unpermeabilized target cells suggests that perforin might be involved in the delivery of granzymes to target cells {Hayes, et al, supra, (1989); Shi, L., et al, (1992), "A natural killer cell granule protein that induces DNA fragmentation and apoptosis", J. Exp. Med., 175: 553-566}. Finally, transfection of RBL cells with a combination of perforin and granzyme A confers the ability to induce DNA fragmentation in selected target cells {Shiver and Henkart, supra, (1991)}. Because the amount of DNA fragmentation induced by these cells is significantly less than that induced by CTLs, it is possible that additional granule-associated molecules are involved in the induction of apoptotic cell death.
Recently, another class of granule-associated proteins that are also able to induce DNA fragmentation in CTL target cells has been identified. TIA-1 is an RNA-binding protein that was initially identified by a monoclonal antibody (2G9) reactive with a 15 kD protein whose expression was restricted to CTLs and NK cells {Anderson, P., et al, (1990), "A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes", J. Imunol., 144: 574-582}. Mitogenic activation induced the expression of immunoreactive isoforms of TIA-1 that migrated at 28 kD, 40 kD and 53 kD. Immunoselection of a .lambda.gt11 cDNA library using the monoclonal antibody reactive with TIA-1 identified two related cDNAs that encode p15-TIA-1 (1T4T8.9-5, 1.6 kb) and p40-TIA-1 (12G9.4, 2.2 kb) {Tian, Q., et al, (1991), "A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells", Cell, 67: 629-639}. Both TIA-1 isoforms were able to induce DNA fragmentation in permeabilized target cells, suggesting that they might be the granule-associated proteins responsible for the induction of apoptotic cell death in CTL target cells. Nothing is known about the molecular mechanisms by which TIA-1 triggers DNA fragmentation in target cells. Identification of cDNAs encoding TIA-1 binding proteins would be a first step in the molecular characterization of TIA-1 function. Further, characterization of the proteins would be useful to screen for drugs that induce apoptotic death in target cells.